To this date, the evaluation of the success or the failure of vasectomy as well as the detection of problems associated with male fertility have been established by performing analyses such as post-vasectomy spermograms or fertility spermograms.
Such analyses typically involve the step of collecting a semen sample from the subject under examination and rapidly analysing the sample (within about 2 hours for vasectomy cases and within about 1 hour for fertility cases).
For current post-vasectomy spermograms a sample of the ejaculate is applied onto a microscope slide or to a hemocytometer for the determination of spermatozoa concentration by microscopic observation. The success of vasectomy is confirmed if a concentration of less than 0.1×106 spermatozoa/mL is observed.
Proof or demonstration of the absence of spermatozoa in a semen sample obtained post-vasectomy may be more complex as an absence of spermatozoa on the microscope slide or on the hemocytometer may be the consequence of actions such as pipetting, loading or miss-observation rather than a direct consequence of the vasectomy procedure.
The reference values confirming the success of a vasectomy are set as <0.1×106 spermatozoa/mL by the American Urological Association (AUA) recommendations or as <10×103 spermatozoa/mL by the European Association of Urology (EAU). It is thus practically impossible to evaluate enough of the sample by microscopy to obtain a decent reproducible value. Visualisation of the entire hemocytometer represents examination of a 0.1 μL sample. For this sample volume, the reference value represents the observation of no more than 10 spermatozoa if the AUA recommendations are followed or of a single spermatozoon if the EAU recommendations are followed, giving rise to variations equal to 10% to 100% of the reference values each time a single cell is observed.
According to the World Health Organization (WHO) (World Health Organization (2010): WHO Laboratory Manual for the Examination and Processing of Human Semen, Fifth edition), the concentration of leukocytes in an ejaculate is to be determined by spermogram analysis. Leukocytes present in the ejaculate can create an oxidative stress that can be detrimental to spermatozoa ability to fertilize or can be an indicator of infection. The ability of the spermatozoa to fertilize as well as the presence of an infection are thus important factors to monitor by spermogram following procedures such as vasectomy and vaso-vasostomy. They are also important parameters in situations where conception is awaited since the presence of leukocytes is associated with production of reactive oxygen species (ROS) in the ejaculate. High ROS concentrations are well known to be detrimental to sperm function.
Determining leukocyte concentration in a semen sample is also problematic because the normal reference values are below 1 million cells per mL (1 M/mL). Consequently, concentrations are based on very few cells. In addition, leukocytes can easily be confounded with immature germ cells due to their similar shape and size.
The WHO suggests a method of microscopic immunofluorescence to confirm the identity of leukocytes in a semen sample. Although this protocol allows for the distinction of leukocytes from other cell types, it is laborious and presents standardization difficulties. Moreover, this protocol does not allow for cell concentration measurements. Even if it could allow for such measurements, the number of cells examined by using such a protocol would still remain very low and would give rise to unacceptable variations in the test results.
Although sperm concentration is a key element of the actual post-vasectomy spermograms, such spermograms are not controlled for the ability to determine sperm concentration in a post-vasectomy sample. The only controls actually available address the ability to confirm the presence or absence of spermatozoa but not spermatozoa concentration.
Both the AUA and the EAU mention that although sperm concentration is a key point, sperm motility is also important. No motile spermatozoa should be observed following a vasectomy no matter what the concentration is. This may however be problematic in that there is a possibility that spermatozoa motility has faded out by the time the semen sample reaches the laboratory for analysis.
There is thus a need in the field of fertility for new and more accurate, quantitative, manageable, controllable and valid methods to evaluate the success of vasectomy and to determine the concentration of spermatozoa in semen samples as well as to assess the integrity, function, activity and motility of spermatozoa in semen samples.